The present invention relates to compositions and methods for template-dependent enzymatic synthesis of nucleic acid, and more specifically, replication of nucleic acid by human polymerase xcex1. The present invention is particularly useful for screening chemotherapeutics for potential mutagenicity and carcinogenicity.
The term xe2x80x9cchemotherapyxe2x80x9d simply means the treatment of disease with chemical substances. The father of chemotherapy, Paul Ehrlich, imagined the perfect chemotherapeutic as a xe2x80x9cmagic bulletxe2x80x9d; such a compound would kill an invading organism without harming the host. This target specificity is sought in all types of chemotherapeutics, including antimicrobial and anticancer agents.
Unquestionably, the greatest success with antimicrobials in terms of specificity has been with antibiotics. The antibiotic penicillin is widely known for its ability to block the synthesis of the cell wall for particular bacteria without interfering with the biochemistry of mammalian cells. What is not widely known is that penicillin is the exception rather than the rule; only a fraction of the thousands of identified antimicrobial drugs are non-toxic to humans.
Efforts to treat viral infection have been largely ineffective for precisely this reason. While a virus is essentially nothing more than nucleic acid surrounded by a lipid-protein envelope, a virus invades a host cell and uses the host cell""s machinery to replicate itself. The latter characteristic makes it especially difficult to find drugs which block viral replication and yet leave intact the ability of the host cell to replicate.
Specificity has also been the major problem with anticancer agents. In the case of anticancer agents, the drug needs to distinguish between host cells that are cancerous and host cells that are not cancerous. The vast bulk of anticancer drugs are indiscriminate at this level. For this reason, only a few types of cancer are appropriate for chemotherapy. Surgery and radiation continue to be the favored types of cancer treatment.
While there has been little success with viral infection and cancer, there is continued hope that drugs can be found or designed with the requisite specificity for the treatment of human afflictions. However, even if compounds can be found that do not have immediate toxicity, exhaustive screening is necessary to ensure that the selected compounds are neither carcinogens nor mutagens.
A mutation is a change in the sequence, number or nature of nucleotide bases in DNA. A certain amount of mutation is normal (and perhaps even necessary) in all organisms. A mutagen is a compound that increases the normal frequency of mutation.
One source of mutation is caused by direct modification of a normal base by a mutagen so as to alter its normal base pairing. Another type of mutation is caused by the incorporation of analogs of the normal nucleotide bases during DNA replication. Still other mutations are caused by the incorporation of additional bases or the loss of bases during replication.
Importantly, not all mutagens will result in carcinogenicity. Nonetheless, all carcinogens are mutagens.
It has proven difficult to directly measure mutagenicity of compounds in higher organisms such as mammals. Mutations are rare and it takes great numbers of organisms before they are seen. Current approaches, therefore, utilize microorganisms such as bacteria.
The most widely used mutagen/carcinogen screening assay is the Ames test. The Ames test utilizes several unique strains of Salmonella typhimurium that are histidine-dependent for growth and that lack the usual DNA repair enzymes. The frequency of normal mutations that render the bacteria independent of histidine (i.e., the frequency of spontaneous revertants) is low. Thus, the test can evaluate the impact of a compound on this revertant frequency.
Since some substances are not mutagenic by themselves but are converted to a mutagen by metabolic activation, the compound to be tested is mixed with the bacteria on agar plates along with a liver extract. The liver extract is needed to mimic metabolic activation in an animal. Control plates have only the bacteria and the extract.
The mixtures are allowed to incubate. Growth of bacteria (if any) is checked by counting colonies. A positive Ames test is one where the number of. colonies on the plates with mixtures containing the compound significantly exceeds the number on the corresponding control plates.
When known carcinogens are screened in this manner with the Ames test, approximately ninety percent are positive. When known noncarcinogens are similarly tested, approximately ninety percent are negative.
For many compounds, the Ames test is quite adequate. These compounds (e.g., pesticides, dyes, etc.) are those thought to cause mutations by direct modification of the chemistry of a normal base. It is believed that this nucleic acid modification chemistry will be the same in the bacteria as in mammalian cells. Thus, the change in the revertant frequency of the bacteria is predictive of mutagenicity in mammals.
The Ames test is, however, not definitive for all chemotherapeutics. Indeed, it may be particularly ill-suited to test nucleotide analogs designed as antiviral and anticancer agents. These agents are designed to be incorporated in the target cell nucleic acid during replication. Unfortunately, they may also be incorporated by normal host cells during normal replication and cause subsequent mutations.
In contrast to nucleic acid modification chemistry, incorporation of nucleotide analogs occurs via the replication machinery. It is known that the bacterial replication machinery is distinctly different from that of mammalian cells. Consequently, there is a concern that there will be a class of nucleotide analogs that will not be incorporated by bacteria but that will be incorporated by normal replicating mammalian cells. These compounds would test negative by the Ames test and yet be mutagenic in mammals.
The present invention relates to compositions and methods for template-dependent enzymatic synthesis of nucleic acid, and more specifically, replication of nucleic acid by human polymerase xcex1. The use of human DNA polymerase a is particularly appropriate for screening chemotherapeutics for potential mutagenicity and carcinogenicity. Unlike current screening approaches, the screening approach of the present invention is predictive of the mutagenicity (if any) of nucleotide analogs in mammals.
The present invention contemplates the over-expression of recombinant human DNA polymerase a that is functional, and yet free of contaminating protein typically associated with human DNA polymerase xcex1 purified by traditional biochemical isolation techniques. The expression of recombinant human DNA polymerasexcex1 of the present invention relies on the construction of a full-length cDNA. This full-length cDNA has been found to generate full-length translation products.
The present invention contemplates the use of recombinant human DNA polymerase a for the screening of chemotherapeutics for potential mutagenicity and carcinogenicity. In one embodiment, recombinant human DNA polymerase a is employed to test for incorporation of analogs of the normal nucleotide bases during DNA replication.
The present invention further contemplates the use of recombinant human DNA polymerase xcex1 to test for the binding of viral proteins. In one embodiment, the present invention contemplates co-infection of cells with two expression vectors, one vector coding for the viral protein of interest and the other vector coding for human DNA polymerase xcex1.
It is not intended that the present invention be limited by the expression system for recombinant human DNA polymerase xcex1. The present invention contemplates all forms and sources of expression systems.